N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Lipid composition of the epidermal gel secretion from the Arabian Gulf catfish (Arius thalassinus Ruppell)

Lipids associated with a threat induced epidermal gel secretion from the catfish, Arius thalassinus, have been analyzed. Phospholipids, neutral lipids and glycolipids are all present and each of these subclasses has been analyzed by thin layer and gas chromatography with a general similarity with membrane lipids being noted. The epidermal gel lipids differed from total liver lipids of the catfish. Fatty acid analysis showed the gel lipid to be rich in the unsaturated fatty acids: oleate (omega 7, C18:1), arachidonate (omega 6, C20:4), and docosahexaenoate (omega 3, C22:6). Some prostaglandins were quantitated in lipid extracts from the epidermal gel.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

Postdoctoral Researchers in the UK: A Snapshot at Factors Affecting Their Research Output

Postdoctoral training is a typical step in the course of an academic career, but very little is known about postdoctoral researchers (PDRs) working in the UK. This study used an online survey to explore, for the first time, relevant environmental factors which may be linked to the research output of PDRs in terms of the number of peer-reviewed articles per year of PDR employment. The findings showed reliable links between the research output and research institutions, time spent as PDR, and parental education, whereas no clear links were observed between PDRs'' output and research area, nationality, gender, number of siblings, or work environment. PDRs based in universities tended to publish, on average, more than the ones based in research centres. PDRs with children tended to stay longer in postdoctoral employment than PDRs without children. Moreover, research output tended to be higher in PDRs with fathers educated at secondary or higher level. The work environment did not affect output directly, but about 1/5 of PDRs were not satisfied with their job or institutional support and about 2/3 of them perceived their job prospects as “difficult”. The results from this exploratory study raise important questions, which need to be addressed in large-scale studies in order to understand (and monitor) how PDRs'' family and work environment interact with their research output—an essential step given the crucial role of PDRs in research and development in the country.     

Flight loss linked to faster molecular evolution in insects

The loss of flight ability has occurred thousands of times independently during insect evolution. Flight loss may be linked to higher molecular evolutionary rates because of reductions in effective population sizes (N_e) and relaxed selective constraints. Reduced dispersal ability increases population subdivision, may decrease geographical range size and increases (sub)population extinction risk, thus leading to an expected reduction in N_e. Additionally, flight loss in birds has been linked to higher molecular rates of energy-related genes, probably owing to relaxed selective constraints on energy metabolism. We tested for an association between insect flight loss and molecular rates through comparative analysis in 49 phylogenetically independent transitions spanning multiple taxa, including moths, flies, beetles, mayflies, stick insects, stoneflies, scorpionflies and caddisflies, using available nuclear and mitochondrial protein-coding DNA sequences. We estimated the rate of molecular evolution of flightless (FL) and related flight-capable lineages by ratios of non-synonymous-to-synonymous substitutions (dN/dS) and overall substitution rates (OSRs). Across multiple instances of flight loss, we show a significant pattern of higher dN/dS ratios and OSRs in FL lineages in mitochondrial but not nuclear genes. These patterns may be explained by relaxed selective constraints in FL ectotherms relating to energy metabolism, possibly in combination with reduced N_e.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Glucoamylase production byAspergillus awamori on rice flour medium and partial characterization of the enzyme

Summary Aspergillus awamori ATCC 22342 was selected from 12 strainsof Aspergillus spp.and Rhizopus spp. as the best producer of amylase. Optimal growth conditions for the enzyme production in shake flasks were provided by: a medium containing 60 g/1 rice flour, 0.075% (w/v) NaNO₂ and 0.075% (v/v) corn-steep liquor, a temperature of 30° C and initial pH value of 6.5. The enzyme was characterized as a glucoamylase with a molecular weight of 49,000. Maximum enzyme activity occurred at 45 C and pH 5.8. The enzyme was stable at 40° C and lost 70 and 90% of activity when heated for 30 min at 50 and 60°C, respectively. Thermal inactivation was slowed in the presence of starch. Michaelis-Menten constants for soluble starch and dextrin were estimated as 12.5 and 33.3 mg/ml, respectively. This enzyme may be used for the production of glucose-rich syrups from rice starch.

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Producción de glucoamilasa por Aspergillus awamori en harina de arroz y caracterización parcial del enzima

Resumen Aspergillus awamori ATCC 22342 se seleccionó entre 12 cepas deAspergillus spp. y deRhizopus spp. como el mejor productor de amilasa. La condiciones óptimas de crecimiento para la producción del enzima en frascos de agitación fueron las siguientes: un medio con la composicion siguiente: 60 g/1 de harina de arroz, 0.075% (m/v) NaNO₂ y 0.075% (v/v) de extracto de maíz (corn steep liquor); una temperatura de 30°C y un pH inicial de 6.5. El enzima fue caracterizado como una glucoamilasa de peso molecular 49,000. La máxima actividad enzimática se obtuvo a 45°C con un pH de 5.8. El enzima era estable a 40 C pero perdió un 70 y un 90% de su actividad cuando se calentó durante 30 min a 50 y 60° C respectivamente. La inactivación térmica fue más lenta en presencia de almidón. Las constantes de Michaelis-Menten para almidón soluble y para dextrina se estimaron como 12.5 y 33.3 mg/ml respectivamente. Este enzima puede utilizarse para la producción de jarabes ricos en glucosa a partir de almidón de arroz.

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Production de glucoamylase par Aspergillus awamori cultivé sur milieu à la farine de riz et caractérisation partielle de l'enzyme

Résumé Aspergillus awamori ATCC 22342 a été sélectionné parmi 12 souches d'Aspergillus spp. et deRhizopus spp. comme étant le meilleur producteur d'amylase. Les conditions optimales de croissance pour la production d'enzyme en fioles agitées sont: un milieu contenant 60 g/1 de farine de riz, 0.075% (w/v) de NaNO₂ et 0.075% (v/v) de liqueur de corn steep, une température de 30° C et un pH initial de 6.5. L'enzyme a été caractérisé comme étant une glucoamylase de poids moléculaire 49,000. L'activité maximum de l'enzyme se situe à 45°C et pH 5.8. L'enzyme est stable à 40°C et perd 70 et 90% de son activité par chauffage pendant 30 min à 50 et à 60°C, respectivement. L'inactivation thermique est ralentie en présence d'amidon. Les constantes de Michaelis-Menten pour l'amidon soluble et pour la dextrine ont été éstimées, respectivement, à 12.5 et 33.3 mg/ml. Cet enzyme peut être utilisé pour la production de sirops riches en glucose à partir d'amidon de riz.

     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

“SIF” cells in the sympathetic ganglia of the bullfrog,Rana catesbeiana: variety in population and innervation

Summary Small intensely fluorescent (SIF) cells appeared singly or, more frequently, in variably-sized clusters in the sacroccygeal 8th and 9th sympathetic ganglia of the bullfrog. Smaller clusters containing only two to nine SIF cells accounted for 61% of 1773 clusters examined. The largest cluster contained 283 cells. The number of cells in individual ganglia also varied from 21 to 3332. SIF cells, solitary as well as in smaller clusters, received no distinct form of the synaptic contact. In contrast, the cells in larger clusters were frequently innervated by nerve endings that were similar in vesicular constitution to the nerve endings on principal ganglion (PG) cells. No synaptic contact was found between SIF cells and PG cells. SIF cells were also characterized by their location in the vicinity of blood capillaries with a continuous endothelium. p]Our observation seems to suggest that larger clusters of SIF cells receiving nerve endings are linked to a paracrine and/or endocrine system. Chemical influence via the blood stream and intraganglionic milieu for non-innervated SIF cells in the solitary or smaller clusters is a subject for speculation. An interneuronal role of SIF cells to relay stimuli to PG cells seems unlikely. The possible functions here assigned to SIF cells could be variable in efficiency depending on their population and density.